Vectors encoding brain derivable polypeptide factors

ABSTRACT

Novel polypeptides, polynucleotide sequences, DNA constructs and compositions are provided for the preparation and use of polypeptides associated with naturally occurring polypeptides found in brains. The low molecular weight polypeptides either are growth inhibitors for neoplastic cells without inhibiting normal cells or affect GABA-ergic transmission. The polypeptides find use in inhibiting neoplastic growth, modulating diazepam receptor response, and detecting receptors for the polypeptides. Antibodies are provided in conjunction with the polypeptides, which may be used together or separately for detecting the presence of the polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Application Ser. No. 823,159, filedJan. 27, 1986, now U.S. Pat. No. 4,963,485 which is continuation-in-partof Application Ser. No. 766,864, filed Aug. 15, 1985, now U.S. Pat. No.4,806,492, which is a continuation-in-part of Application Ser. No.694,712, filed Jan. 25, 1985, now U.S. Pat. No. 4,714,683, issued Dec.22, 1987, which disclosures are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Cellular growth and differentiation appear to be initiated, promoted,maintained and regulated by a multiplicity of stimulatory, inhibitoryand synergistic hormones and factors. The alteration and/or breakdown ofthe cellular homeostasis mechanism seems to be a basic cause of growthrelated diseases including neoplasia. There is a considerable interestin the isolation, characterization and mechanism of action of growthmodulatory factors (stimulators and inhibitors) because of theirpotential use in the diagnosis, prognosis and therapy of variousdiseases, such as cancer, as well as in understanding the basicmechanisms of mitosis, particularly as it may affect cancer.

Besides growth and differentiation, many bodily responses are regulatedby proteins, where the proteins may serve as ligands or receptors.Further investigation of the regulation of brain function and responseto external and internal stimuli has resulted in the isolation of amyriad of compounds which are involved in the regulation of responses tosuch stimuli as pain, mood, or the like.

Benzodiazepines (BZD), commonly used as anxiolytics, anticonvulsants,muscle-relaxants and sedatives, are believed to exert theirpharmacological effects based on the potentiation of the γ-aminobutyricacid (GABA)-mediated inhibitory neurotransmission. The first step in themodulation of GABA-ergic transmission by BZD appears to be binding tospecific high affinity and saturable binding sites in the centralnervous system, where the binding sites are believed to be a componentof a "supramolecular complex." The need to understand this system, aswell as being able to modulate or control the system is dependent onknowing the naturally occurring ligand and the manner in which itfunctions.

The detection, isolation and purification of these factors is frequentlycomplicated by the complexity of the mixture, the divergencies ofactivities of the various components in the mixtures, the sensitivity ofcomponents to deactivation by a wide variety of reagents, the potentialfor having compounds which depend for their activity on the presence ofmultiple subunits, and the frequent difficulties in providing bioassaysfor tracking various purification steps. Nevertheless, there have beensubstantial advances in purification and separation, which advances haveaided in the detection and isolation of products of interest.

2. Description of the Prior Art

Beal et al., Cancer Biochem. Biophys. (1979) 3:93-96 report the presenceof peptides in human urine which inhibit growth and DNA synthesis morein transformed cells than in normal cells. Holley et al., Proc. Natl.Acad. Sci. (1980) 77:5989-5992 describe the purification of epithelialcell growth inhibitors. Letansky, Biosci. Rep. (1982) 2:39-45 reportthat peptides purified from bovine placenta inhibit tumor growth andthymidine incorporation in DNA to a greater extent in neoplasms than innormal cells. Chen, Trends Biochem. Sci. (1982) 7:364-365 reports theisolating of a peptide from ascites fluid with a cancer suppressingproperty. Redding and Schally, Proc. Natl. Acad. Sci. (1982)79:7014-7018 report isolation of purified peptide(s) from porcinehypothalmi which exhibit antimitogenic activity against several normaland tumor cell lines. Most of these factors have not been fullycharacterized, nor are their primary structures known.

Diazepam binding inhibitor (DBI) is reported and described by Guidotti,et al., Proc. Natl. Acad. Sci. USA (1983) 30:3531-3525, Costa, et al.,Neuropharmacol. (1984) 23:989-991, Ferrero, et al., ibid (1984)23:1359-1362, and Alho, et al., Science (1985) 229:179-182.

SUMMARY OF THE INVENTION

Novel methods and compositions are provided which compositions areisolatable from brain tissue using acidic aqueous acetonitrile on a gelpermeation column followed by reverse phase HPLC employing a lineargradient of acidic (0.1% trifluoroacetic acid) aqueous acetonitrile, aswell as the components of such composition and polypeptides havingregions of substantial homology with such components. The compositionsand components may find use in retarding growth of neoplastic cells, asagonists for diazepam and as surface membrane proteins. Polynucleotidesequences are provided encoding the polypeptides, which allow forproduction of the polypeptides in prokaryotes or eukaryotes. Antibodiesare provided which bind specifically to the subject polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the nucleotide sequence and deduced amino acid sequenceof a CDNA clone derived from bovine brain (*). Amino acid residues arenumbered relative to the proposed initiating methionine. The regionhomologous to EBZD is overlined by a heavy line, and extended regions ofhydrophobic and uncharged amino acids are delineated by lighter lines.Potential glycosylation sites are boxed.

FIG. 2 indicates the human and bovine DNA sequences.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel compositions are provided comprising combinations of naturallyoccurring physiologically active polypeptide compounds, the individualpolypeptide compounds, physiologically active fragments thereof, andnucleic acid sequences encoding the various polypeptide compounds. Thepolypeptides have sequences which are homologous or substantiallyhomologous (at least 60% equivalency) to polypeptides found naturally inbrain cells of vertebrates, particularly mammals. The compositions havediverse physiological activity and may be specifically localized inbrain cells or be found generally in a wide variety of tissues otherthan brain. The polypeptides are characterized as being isolatable byelution from a chromatographic column employing homogenized brain tissueor purified fraction thereof with an acidic aqueous acetonitrile eluent.One factor can be isolated substantially pure by chromatography usingreverse phase HPLC and a slow linear gradient of acidic aqueousacetonitrile. For the other component the desired activity may befurther purified using aqueous acidic n-propanol as a linear gradient.The chromatogram may be developed over a period of 4hr or more in orderto obtain reasonable separation with flow rates of about 0.1-0.5ml/min.The separation employing the rpHPLC will usually be with a partiallypurified sample from brain tissue. The partial purification may beconveniently achieved employing gel permeation chromatography withacidic aqueous acetonitrile as the eluent, particularly elutingisocratically. Conveniently, the media may be 40% acetonitrile in 0.1%aqueous trifluoroacetic acid.

The two components have substantially different physiological activitiesand composition. One factor which will be referred to as endogenousbenzodiazepineoid or endozepine (EBZD), shows activity as a valiumagonist, binding to diazepam receptors and appears to be a cytoplasmicprotein. At low dosage, the EBZD causes agitation and heightenedawareness in mammals, while at high dosage, causes sedation. Withbeating heart cell aggregates, low doses slow the heart beat rate, whilehigh dosages increase the heart beat rate. The compounds show somehomology with the sequence published for diazepam binding inhibitor(Guidotti et al., supra).

The peptides are further characterized by having a K_(i) of about1-10μM, usually about 3-6μM, in inhibiting the binding of benzodiazepineto brain synaptosomes (see Experimental). The naturally occurringpolypeptides will have molecular weights in the range of about 7 to15kDal, usually 8 to 12kDal, as measured by gel permeationchromatography or 6 to 10kDal with SDS-PAGE (see Experimental). Thepeptides are hydrophilic. Specific activities can be obtained whichexceed 1000 units/mg (see Experimental).

Fragments of EBZD are also bioactive; particularly fragments includingamino acids 33 to 52 of EBZD (consensus sequence among differentspecies) have activity as a diazepam binding inhibitor.

The second component is referred to as brain factor (BF) and can beisolated from brain tissue as indicated above. This factor is present insubstantially smaller amount than the EBZD and lags EBZD in the gelpermeation purification. The compound is found to be effective ininhibiting growth of lung carcinoma cells, as well as inhibiting softagar colony formation and plating efficiency, all as described in theExperimental section.

BF will generally have a molecular weight of about 5 to 20kDal, usuallyabout 8 to 18kDal, as determined from gel permeation chromatography. Theproteins can be purified using rpHPLC and a linear gradient 0.1% TFAaqueous n-propanol eluting at about 20 to 26% n-propanol. The purifiedprotein can have a specific activity of at least about 15×10³ units/μg(see Experimental).

A third component may be obtained by using probes which are a pool ofoligonucleotides which encode for the amino acid sequence KWDAWN, theamino acids 54 to 59 of hEBZD. The protein is substantially larger thanEBZD, has a leader sequence and a stop transfer sequence, so as to becapable of acting as a surface membrane protein, where the major portionof the peptide is external to the cell. This protein is substantiallylocalized in brain tissue.

The proteins may be obtained from any convenient source, particularlyvertebrates, more particularly mammals, including bovine, ovine,lagomorpha, murine, primate, particularly human, and the like. Thecompounds may be used as they occur naturally or may be modified invarious ways, such as lacking glycosylation, lacking terminal acylation,particularly acetylation or formylation, employing fragments havingphysiological activity, such as competitive binding capability to areceptor, e.g., an antibody, having deletions, insertions, being fusedto other peptides, being covalently joined to other peptides, e.g.,immunogens for antibody formation, or haptens, or being mutated byhaving one or more, usually not more than about 10 number percent, moreusually not more than about 5 number percent, of the amino acids beingvaried, by insertions, deletions, transitions or transversions.

Each of the polypeptides will now be considered in further detail.

EBZD Polypeptides

The EBZ polypeptides are characterized by being of less than about 20kilodaltons (kDal), usually less than about 15kDal and will usually beat least about lkDal, more usually at least about 2kDal.

The polypeptide compositions are further characterized by eluting from aμ-Bondapak-C₁₈ column in reverse phase HPLC under ambient conditionsemploying a linear gradient of 0-60% acetonitrile in 0.1% aqueoustrifluoracetic acid in the range of 28-50% acetonitrile, particularly29-40% acetonitrile, and more particularly about 29-36% acetonitrile,more specifically 30-34% acetonitrile.

The polypeptides will have at least about 15 amino acids, more usuallyat least about 20 amino acids, and fewer than about 125 amino acids,usually fewer than about 100 amino acids. The naturally occurringcompounds will have a molecular weight in the range of about 7 to 15kDalusing PAGE or gel permeation chromatography as described in theExperimental section.

The polypeptide composition will be further characterized by having atleast one of the following amino acid sequences, preferably at least twoof the following amino acid sequences, and more preferably at leastthree of the following amino acid sequences, where the sequence may beconserved by the insertion or deletion of up to and including threeamino acids or combination thereof.

    ______________________________________                                        a. Y aa.sup.e aa.sup.a Ar K A T aa.sup.b                                      b. K W D A W                                                                  c. A M aa.sup.c A Y (X).sub.x V E E                                           d. T K P aa.sup.d aa.sup.p E E M L F I Y aa.sup.e H Y K                       e. Q A T V G D I N T E R P G M L D                                            f. Q A T V G D l N T E R P G M L D F T G K                                    g. K G T S K E D A                                                            ______________________________________                                    

wherein:

aa^(a) is an aromatic amino acid, particularly phenylalanine andhistidine;

aa^(b) is any amino acid, particularly an aliphatic amino acid, whichmay be acidic, basic, or neutral, preferably acidic or neutral of fromabout 3 to 5 carbon atoms;

aa^(c) may be any amino acid, particularly an aliphatic amino acid,which is basic, acidic or neutral, more particularly basic or neutral offrom about 5 to 6 carbon atoms;

aa^(d) is an aliphatic neutral amino acid, which may be polar ornon-polar, particularly having an hydroxyl substituent and of from about3 to 4 carbon atoms;

aa^(e) is an aliphatic neutral amino acid, which may be polar ornon-polar, particularly having an hydroxyl substituent and of from about2 to 4 carbon atoms;

aa^(p) is an aliphatic amino acid, which may be neutral polar or acidic,particularly acidic or the amide thereof, and of from 4 to 5 carbonatoms;

Ar is an aromatic amino acid, including tyrosine, phenylalanine,histidine and tryptophan, particularly Y;

aa^(q) is an aliphatic amino acid, particularly a basic or neutralpolar, amino acid of from 4 to 6 carbon atoms, when neutral polarparticularly of from 4 to 5 carbon atoms having an amide group, i.e., Nand Q, preferably K;

X is from 1 to 3 amino acids, which may be any amino acids, particularlyaliphatic amino acids, more particularly having a first neutral aminoacid, a second acidic amino acid or amide thereof, and a third basicamino acid; and

x is 0 or 1.

For the purposes of the subject invention, the various amino acids willbe divided into a number of subclasses. The following Table indicatesthe subclasses:

    ______________________________________                                        aliphatic                                                                     neutral                                                                       non-polar           G A P V L I                                               polar               S T C M N Q                                               acidic              D E                                                       basic               K R                                                       aromatic            F H Y W                                                   ______________________________________                                    

Of particular interest are polypeptides having the above physiologicalcharacteristics and including at least one of the following amino acidsequences:

    ______________________________________                                        a. TKPaa.sup.d DEEMLFIYaa.sup.e HYKQATaa.sup.f G                              b. KWADAWaa.sup.g aa.sup.h Laa.sup.i aa.sup.j aa.sup.k KEaa.sup.m             AMaa.sup.n AY(X).sub.x VEEaa.sup.o KK                                         c. KQATVGDINTERPGMLDFT                                                        ______________________________________                                    

wherein:

aa^(d), aa^(e), aa^(q), and Ar are as defined previously;

aa^(f) is an aliphatic amino acid, which may be neutral or acidic,particularly of from about 4 to 6, more particularly neutral, of from 5to 6 carbon atoms;

aa^(g) is an aliphatic neutral amino acid, particularly a polar aminoacid of from about 3 to 5, more usually of from 3 to 4 carbon atoms,particularly having an hydroxyl or carboxamido polar substituent;

aa^(h) is an aliphatic amino acid, which may be neutral or acidic,particularly polar or acidic, having an hydroxyl substituent and of fromabout 3 to 5 carbon atoms;

aa^(i) is an aliphatic amino acid, particularly a neutral or basic aminoacid, more particularly a non-polar neutral amino acid, of from about 2to 6 carbon atoms;

aa^(j) is an aliphatic amino acid, either neutral or acidic, whenneutral, preferably non-polar, and particularly of from about 2 to 5,more usually of from 2 to 4 carbon atoms;

aa^(k) is an aliphatic neutral amino acid, particularly a polar aminoacid of from about 3 to 5, more usually of from 4 to 5 carbon atoms,having a chalcogen (oxygen or sulfur) functionality, particularlyhydroxyl or methylthio;

aa^(l) is an aliphatic neutral amino acid, particularly a polar aminoacid, having an hydroxyl functionality and of from about 3 to 4 carbonatoms;

aa^(m) is an aliphatic acidic amino acid of from 4 to 5 carbon atoms;

aa^(n) is an aliphatic neutral or basic amino acid, of from about 3 to 6carbon atoms, particularly of from 4 to 6 carbon atoms, where thealiphatic neutral amino acid is preferably non-polar;

aa^(r) is an aliphatic neutral non-polar or polar amino acid,particularly non-polar of from 3 to 6 carbon atoms, preferably A;

aa^(s) is an aliphatic neutral non-polar or polar amino acid of from 3to 4 carbon atoms, when polar, particularly having an hydroxy group,preferably A;

X' is the same as X, usually from 1 to 3 amino acids which are aliphaticamino acids, which may be neutral, acidic or basic, generally of from 4to 6 carbon atoms, particularly in the order in the N-C directionneutral non-polar, acidic or amide thereof, and basic;

x is 0 or 1; and

aa^(o) is an aliphatic neutral amino acid, which may be polar ornon-polar, particularly of from about 4 to 6, usually 5 to 6 carbonatoms, when polar, particularly having a methylthio group.

It being understood, that besides the X, there may be from 1 to 3,preferably 1 to 2, insertions or deletions to maintain the consensusstructure between the various members of the family of polypeptides ofthis invention. Also, the amino acids are the naturally-occurringL-amino acids, although in some instances the D-amino acid may find use.

Compounds of particular interest have the following formula or fragmentthereof of at least 10, usually at least 15 and preferably at least 25,amino acids coming within the following sequence, particularly fragmentsincluding one of the prior specified sequences: ##STR1## wherein: φ is Hor acetyl;

Z^(N) is a bond or from 1 to 10 amino acids, preferably from 1 to 9amino acids, which may be aliphatic or aromatic, where the sequence isselected from the sequence ##STR2## so that any sequence of amino acidswithin such sequence may be joined to aa^(l), for example E-A-A orH-E-T-R, or the like, where one may select either amino acid where twoamino acids appear at the same site, preferably the amino acids on thesame line being taken together;

aa⁴,8,11 are aliphatic neutral non-polar amino acids of from 2 to 6carbon atoms, particularly glycine, alanine, proline, valine, leucineand isoleucine;

aa⁵,16,25,35,37,55,60,61,68,73,79 are aliphatic neutral non-polar orpolar amino acids, where particularly aa⁵,16,25,35,37,73,79 are either,while the remainder are preferably polar amino acids, particularlyserine, threonine, cysteine, methionine, asparagine and glutamine;

aa¹,6,7,17,19,32,34,38,56,59,64,78 are aliphatic neutral amino acids,including polar and non-polar amino acids or aliphatic acidic aminoacids, i.e., aspartic and glutamic amino acids; particularly,aa¹,7,32,34,59,78 are non-polar when neutral amino acids with theremainder being polar when neutral amino acids, andaa¹,6,7,17,19,34,38,56,64 are preferably acidic amino acids;

aa²,10,13,23,25,26,27,42,43,45,49,77 are aliphatic neutral amino acidsor aromatic amino acids, particularly phenylalanine, histidine, tyrosineand tryptophan, preferably aa²,10,26,27,45,77 are preferably aromaticamino acids, while the remaining amino acids are preferably aliphaticamino acids;

aa³,9,12,14,36,39,46,48,58,67,76 are aliphatic neutral or basic aminoacids, i.e., lysine and arginine, where aa³,58,67,76 are preferablynon-polar when other than basic and the remainder are preferably polarwhen other than basic, except for aa⁴⁶ which may be either polar ornon-polar, and aa³,12,14,39,48,58,67,76 are preferably basic aminoacids;

aa²² is basic or aromatic, particularly phenylalanine;

X and x have been defined previously; and

Z^(C) is OH, NH², or a sequence of from 1 to 6, usually 1 to 4 aminoacids, particularly aliphatic amino acids of from 2 to 6, usually offrom 4 to 6 amino acids, particularly polar or non-polar amino acids,particularly a sequence within the sequence M-P-M-T.

It is understood that one or more of the consensus amino acids may bechanged, usually not involving more than two changes, and the consensussequence may require the insertion or deletion of up to 3, preferablynot more than 2, amino acids other than indicated as X.

Polypeptides of interest will have at least 15, preferably at least 30amino acids in a sequence included in the following sequence: ##STR3##wherein any amino acid at any site may be substituted for any otheramino acid at that site, preferably amino acids above are takentogether, while amino acids below are taken together, more preferablyamino acids on the same line are taken together, and an asterisk (*)intends a bond (no amino acid at that site), and z is 0 or 1. Thesequence may extended by up to a total of 10, usually up to a total of8, amino acids, where the N-terminus may have the additional sequenceV-H-E-T-R or any portion thereof and the C-terminus may have thesequence M-P-M-T, or any portion thereof.

Polypeptides of particular interest include polypeptides having at leastabout 15, preferably at least about 20, more preferably at least about30 amino acids, included in one of the following sequences, where suchsequences include at least 10, preferably at least 12, and morepreferably at least 15 of the amino acids indicated with an asterisk (*)(b=bovine, h=human). ##STR4##

For particularly preferred compositions of the subject invention, theabove sequences may not be changed by having more than about 5 aminoacids, inserted, deleted, or substituted, or combinations thereof,preferably not more than about 3 amino acids.

The EBZD compositions will be at least about 20% pure, more usually atleast about 30% pure, and preferably at least about 80% pure, morepreferably at least about 95% pure, desirably 100% pure. Of particularinterest are EBZD compositions which are substantially free ofcomponents from source tissue, having less than 1%, preferably less thanabout 0.1%, and more preferably less than about 0.001% by weight ofcomponents from the source tissue. The subject EBZD compositions willhave a specific activity of at least 15 units/mg as measured by theamount needed for 40% competition of 3H-diazepam binding to crudesynaptosomic membrane under the assay conditions described in theExperimental. Usually, the specific activity will be at least 500units/mg, preferably at least about 1000 units/mg, and may be 1250units/mg or higher. Generally, there will be at least about 10μgequivalent per gram of wet tissue in the pineal body, choroid plexus,pons, and optic nerve, and usually at least about 20μg equivalent pergram of wet tissue in the choroid plexus. As indicated previously, thesubject compounds will be able to affect heart beat rate in beatingheart cell aggregates, slowing the heart beat rate at low doses,generally the low doses being less than about 150μg/ml (see Experimentalsection). Other characteristics of the EBZD will be evident from thevarious tests for which physiological activity is demonstrated for EBZD.

Brain Factor

The brain factor is characterized by being at least 2, more usually atleast about 5, and not more than about 25, more usually not more thanabout 22kDal. The brain factor will have a specific activity asevidenced by the cell growth modulatory assay of at least about 1×103,more usually at least about 5×103, preferably at least about 10⁶,preferably at least about 1.5×10⁷. The brain factor will usually be atleast about 10% pure, more usually at least about 50% pure, preferablyat least about 80% pure, and more preferably at least about 95% pure,desirably 100% pure. Particularly, it will be desirable that the brainfactor be substantially free of components from the tissue from which itis isolated, such as cell debris, other proteins, saccharides, lipids,combinations thereof, and the like.

The brain factor sequence will include a sequence having substantiallythe following amino acid sequence proximal or at the N-terminus:

    N-K-E-L-D-P-V-Q-K-L-F-V-D-K-I-X-E-Y

wherein X indicates any amino acid.

The brain factor suppresses human T-cell responses, so as to find use asan immunomodulator for the treatment of hosts with autoimmunity or organtransplants, being used alone or in conjunction with otherimmunosuppressants, such as cyclosporin A or corticosteroids.

Usually, the brain factor sequence will be at least 65 number percent,preferably at least about 75 number percent homologous to the abovesequence (by indicating number percent, it is intended that deletions,insertions, and mutations be counted as additive differences).

The subJect brain factors will also find activity in inhibiting cellgrowth and inhibiting soft agar colony formation. Thus, the subjectcompounds find use in inhibiting in vitro and in vivo the multiplicationof neoplastic cells, while having substantially less effect on normalcells, usually being able to differentiate by at least one order ofmagnitude, preferably at least two orders of magnitude betweenneoplastic and normal cells.

Membrane Protein

The membrane protein will for the most part have the sequence shown inFIG. 1:

The peptides will therefore have at least about 60%, more usually atleast about 75%, and preferably at least about 95 number percent of thesequence in FIG. 1. Of particular interest is the sequence underlinedwith the heavy dark line which has homology with EBZD. The protein isfurther characterized by being at least about 10% pure, preferably atleast about 50% pure, more preferably at least about 80%, andparticularly preferred at least about 95% pure, desirably pure. Thecompound is desirably free of components from the tissue from which itmay be derived.

The membrane protein is further characterized by being primarily foundin brain tissue.

Additional proteins of interest are included as fragments of the aboveprotein which include polypeptides beginning at the methionine atnucleotide 170 or at nucleotide 270 or 273.

The subject polypeptides may be used as antigens for the production ofantibodies, which in turn may be used as antigens for the production ofanti-idiotypic antibodies. Either polyclonal or monoclonal antibodiesmay be prepared in accordance with conventional ways. The subjectpolypeptides or fragments thereof, generally fragments having at leastabout 15 amino acids, may be used by themselves, but are preferablybound or linked to an adjuvant or antigen which activates the immunesystem. Various antigens may be used, such as serum albumins, keyholelimpet hemocyanin, globulins, or the like. A wide variety of techniquesare available for linking to polypeptides, such as glutaraldehyde,maleimidobenzoic acid, diazobenzoic acid, or the like. Adjuvants includeFreund's adjuvant, aluminum hydroxide, or the like. The antigen isinjected into an appropriate host in conventional amounts, where one ormore booster injections may be made in from 2 to 4 week intervals. Wheremonoclonal antibodies are employed, normally a mouse is injected withthe original and booster injections and the spleen isolated and thesplenocytes fused with an appropriate fusion partner in accordance withconventional techniques. See, for example, Galfre et al., Nature (1977)266:550; Kennett et al., Current Topics in Microbiology and Immunology(1978) 81:77; U.S. Pat. Nos. 4,381,292 and 4,363,799. However, forspecial purposes, other mammals may be employed, such as primates, e.g.,humans, for production of antibodies having human F_(c) chains.

The EBZD polypeptides and antibodies which bind specifically to the EBZDpolypeptides may find use individually or together, both in vivo and invitro. The subject compounds may be used as agonists for binding todiazepam receptors. The subject compounds may be used to determine thenumber and distribution of diazepam receptors. In addition, the subjectcompounds may find use in regulating the heart beat of a mammalian host,for sedating a mammalian host or for causing agitation in a mammalianhost.

The brain factor polypeptides and antibodies which bind specifically tothe brain factor polypeptides may find use individually or together,both in vivo and in vitro. Because of the tumor-inhibiting property ofthe polypeptides, the polypeptides may be used with mixtures of cells,including both normal and tumorigenic cells, to inhibit the overgrowthof the tumorigenic cells. The subject compounds can therefore beemployed in such situations as when mutagenizing normal cells, where onewishes to distinguish between the differences occurring with normalcells and tumorigenic cells as a result of mutagenesis; inhibitinggrowth of tumorigenic cells where bone marrow has been removed from amammalian host; detecting binding sites for the subject polypeptides ina cell population; and the like. The brain factor polypeptides may beused for distinguishing or removing T-cells from other cells.

The brain factor polypeptides may be used in vivo for inhibiting tumorcell growth, by injection into tumors, encapsulation into liposomes,where the liposomes may be bound to antibodies specific for orpreferential for tumor cells, or the like. Alternatively, the subjectpolypeptides may be used as immunomodulators.

The polypeptides may be used by themselves or in combination with theantibodies in diagnosis for the polypeptides. Either or both may belabeled or unlabeled for use in diagnostic assays. A large number ofdiagnostic assays are described in the literature and include thebinding, either directly or indirectly, to the subject polypeptides orantibodies of a variety of labels, such as enzymes, radionuclides,fluorescers, substrates, coenzymes, particles, e.g., magnetic particles,or the like. As illustrative of these assays, see for example, U.S. Pat.Nos. 3,817,837; 3,850,752; 4,174,384; 4,277,437 and 4,374,925.

Various assays are divided arbitrarily into homogeneous andheterogeneous immunoassays, where the distinction is based on whetherthe complex between the polypeptide and its antibody must be separatedfrom the uncomplexed members of the specific binding pair. Variousassays are referred to as EIA, ELISA, RIA, homogeneous EIA, dot-blot,Westerns, or the like.

Antibodies to the subject polypeptides may be used in themselves asantigens to produce anti-idiotypes, which may serve as competitiveantigens, having epitopic sites competitive with epitopic sites of thesubject polypeptides. These anti-idiotypes may therefore serve asdiazepam agonists, tumor inhibitors as substitutes for the subjectpolypeptides or as antagonists for the subject polypeptides.

The above described polypeptides form families of naturally occurringpolypeptides which may be derived from natural sources, as well asnon-naturally occurring polypeptides which share physiologicalproperties, such as binding specificity, e.g., diazepam receptor andtumor cell inhibition. The naturally occurring compounds may be obtainedfrom naturally occurring sources, primarily brain tissue. However, thesubject naturally-occurring compositions may be found in a number ofdifferent tissues, such as spleen and testes.

To isolate the subject compounds, brain tissue is freed of blood clots,homogenized, extracted with an organic solvent under acidic conditions(pH<4) and then dialyzed to remove low molecular weight material, thatis material less than about 4kDal. The resulting product is then freedof compounds which apparently act as inhibitors of its activity using agel permeation column and an aqueous 0.1% trifluoracetic acid solution-containing from about 35 to 45% acetonitrile eluant. The fractions aremonitored by bioassay. The resulting EBZD fraction is further purifiedemploying a reverse phase high pressure liquid chromatographic column,for example, a μ-Bondapak-C18 column employing linear gradients ofacetonitrile of from about 0 to 60% in aqueous 0.1% trifluoracetic acid,and using an extended column and slow rates of elution, e.g., greaterthan 4hr, preferably greater than 5hr. The subject EBZD compounds arefound in the fractions of from about 30 to 50% acetonitrile, moreparticularly from 30 to 40% acetonitrile.

The BF compound containing fraction is further purified using rpHPLCwith a linear gradient of 0.1% aqueous acetonitrile, eluting at about32-36% acetonitrile, with the process being repeated one or more times.This concentrated fraction is then further purified using rpHPLC with alinear gradient of 0.1% aqueous n-propanol, eluting at about 22-24%-n-propanol, with this process being repeated one or more times.

Alternatively, the subject polypeptides may be synthesized in accordancewith known techniques, particularly where the polypeptides are fewerthan 30 amino acids, more particularly fewer than 25 amino acids. See,for example, Barany and Merrifield, Solid-Phase Peptide Synthesis, "ThePeptides, Analysis Synthesis Biology," Special Methods in PeptideSynthesis, Part A, Vol. 2, Gross and Merenhofer, eds., Academic Press,N.Y. 1980, pp. 1-284.

For relatively large polypeptides, particularly those of about 20 aminoacids or greater, more particularly of about 30 amino acids, hybrid DNAtechnology may be employed for obtaining sequences encoding for thepolypeptide, which may then be used for expression of the desiredpolypeptide in accordance with known ways. Genomic DNA, cDNA, syntheticDNA or combinations thereof may be employed for coding for thepolypeptides, the presence of any introns being accommodated byemploying a host cell having a functioning splicing system for theintrons. For the most part, an open reading frame will be employed (freeof introns), where the sequence coding for the open reading frame willbe joined to transcriptional and translational regulatory signals whichare functional in the expression host.

cDNAs of particular interest include the CDNAs obtained for EBZDs. ThecDNA may include up to about 120bp upstream from the initiatingmethionine and about 225bp downstream from the translation terminationsignal, including a poly(A)+tail.

FIG. 2 provides a comparison of nucleotide and deduced amino acidsequences of bovine and human EBZD. Residues that differ in the humansequence are indicated above and below the bovine sequence, which wasdetermined from three overlapping cDNA clones. Nucleotide residues arenumbered relative to the composite sequence of the bovine clones; aminoacid residues are numbered relative to the initiating methionine. Theopen reading frame of the bovine sequence is flanked by asterisks (*).Differences in the poly(A)+addition sites of three separate clones areindicated by an inverted slash. The NaeI and HindIII restriction sitesused for preparing the bovine cDNA probe are indicated.

The sequences provided in the experimental section, or fragmentsthereof, fragments having at least about 45 bases (15 codons or greater)may be employed for expression of polypeptides of the subject invention.By employing in vitro mutagenesis, mutagenesis, adaptors or the like,the sequences can be varied from the naturally-occurring sequence toproduce sequences having silent mutations or codons that code fornon-wild type amino acids. Thus, one can produce bothnaturally-occurring polypeptides and polypeptides having analogousphysiological properties but differing in one or more amino acids.

The coding sequence which is employed may have blunt or cohesive endsfor joining to other sequences. For expression, a large number ofexpression vectors are either commercially available or have beendescribed in the literature. Thus, one can introduce the subjectsequence into an expression vector for expression in an appropriatehost. The hosts may be prokaryotic or eukaryotic, that is, bacteria,algae, fungi, e.g., yeasts, mammalian cells, e.g., mouse cells, hamstercells, monkey cells, or the like. The expression vector, whethercompletely assembled for insertion of the coding sequence or assembledin conjunction with the coding sequence for a polypeptide of the subjectinvention, will be characterized for the most part as follows. Usually,but not always, a replication system other than the wild-typereplication system will be available, providing for a low or high copynumber of an episomal element to be maintained in the host. Whereintegration of the coding sequence into the host genome is desired, thereplication system will not be required. The coding sequence will beflanked at the 5' and 3' ends by transcriptional and translationalinitiation and termination regulatory signals respectively, frequentlyother than the wild-type regulatory regions. Therefore, promoter regionswill be employed at the 5' end, which may include capping sequences,operators for regulated expression, the absence of promoter regulationfor constitutive expression, enhancer sequences, and the like. At the 3'terminus will be a terminator, stop codons, optimally a polyadenylationsequence, and the like. The expression construct of the transcriptionaland translational regulatory sequences and coding sequence willfrequently be joined to one or more markers which allow for selectionboth as to the transformed hosts into which the vector has beenintroduced and providing for a competitive advantage for those cellswhich retain the expression vector. Markers may include complementationby providing prototrophy to an auxotrophic host, biocide resistance,such as resistance to various antibiotics, heavy metals, or the like,immunity, etc. The various sequences will be selected so as to befunctional in the host.

With many expression vectors, a polylinker is present between thetranscriptional and translational initiation and termination regulatorysequences which provide for a plurality of restriction sites. Thus, byappropriate design of the coding sequence or the use of adaptors, thecoding sequence may be inserted into the polylinker region.

In certain situations, it may be desirable to join the coding sequenceto a 5'-coding sequence so as to obtain a fused product, where the5'-sequence codes for a leader sequence, particularly for a secretoryleader sequence and processing signal. Various secretory leaders havebeen described in the literature; see, for example, U.S. Pat. No.4,411,994, EPA 88,632 and 116,201. The coding sequence is joined inproper reading frame to the secretory leader and processing signal,whereby the fused coding sequence may then be inserted into theexpression vector to provide for the expression construct.

Where the polypeptide is retained intracellularly, after growing thecells, the cells may be harvested and lysed and the polypeptideisolated. Where the polypeptide is secreted, the nutrient medium may becontinuously exchanged and the polypeptide isolated. Various techniquesexist for isolation and purification of polypeptides, such as affinitychromatography, HPLC, electrophoresis, gradient centrifugation, solventextraction, and the like.

Depending upon the use, the subject compounds may be formulated in avariety of ways. For in vivo administration, the subject compounds maybe introduced into a physiologically acceptable carrier, such as sterilewater, saline, phosphate-buffered saline, ethanol, etc. Theconcentrations will vary widely depending upon the particularapplication, whether the application is localized or general, or thelike. Administration may be parenterally, intravenously,intraperitoneally, intra-arterially, subcutaneously, etc.

For the naturally occurring EBZDs, concentrations will generally be fromabout 5 to 500μg/mL. Depending upon the manner of administration,dosages may vary from about 0.5 to 500μg/kg, more usually from about 5to 50μg/kg, with doses exceeding about 25μg/kg having tranquilizingeffects. The particular dosage will vary with the desired response, themanner of administration, the repetitive nature of the dosages, and thelike.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Materials and Methods Materials

Bio-Sil TSK-250 gel filtration HPLC columns were purchased from Bio-RadLaboratories, Richmond, CA. μ-Bondapak-C18 columns were bought fromWaters Associates, Milford, MA. Trypsin (TPCK treated), chymotrypsin andStaphylococcol aureus V8 protease were from Worthington. Theendoproteinase Lys-C was from Boehringer-Mannheim. ³ H-labeled diazepam,R015-1788, flunitrazepam, and β-carboline were obtained from NEN,Boston, MA. Carrier free ¹²⁵ I-iodine was from Amersham, ArlingtonHeights, IL.

Purification of Endo-benzodiazepineoid (EBZD) from Bovine Brain

Fresh or frozen bovine brain (480g wet weight) was thawed and minced.The minced tissue was suspended in 2400ml and extraction bufferconsisting of 2379ml ethanol (98%), 18.5ml concentrated HCl, 84mgphenylmethylsulfonyl fluoride and 2.5ml of aprotinin [23 TIU/ml frombovine lung (Sigma Chemical Co.)]. The mixture was homogenized in aWaring commercial blender. The homogenate was stirred at 4° C.overnight, centrifuged at 8,000rpm in a Beckman type 19 rotor for 30min,and the supernatant carefully removed. The final volume was about2070ml. Chloroform (2070ml) and 207ml of acidified water (203ml waterand 4ml concentrated HCl) was added to the supernatant, the mixturestirred vigorously for approximately 1h, and allowed to stand at roomtemperature to separate into two phases. The upper aqueous phase wascarefully removed and dialyzed against 20 liters×2 changes of 0.1Macetic acid at 4° C. in a Spectrapor dialysis membrane tubing (cut-off3,500MW, American Scientific Products). The dialyzed supernatant waslyophilized. The lyophilized material (785mg) was termed crude fraction.

Gel Permeation Chromatography

A Bio-Sil TSK-250 column (60×2.1 cm) was attached to a high pressureliquid chromatography (HPLC) system (Waters Associates). The crudefraction was dissolved in 40% acetonitrile in water with 0.1%trifluoroacetic acid at a concentration of 8mg/ml. The column wasequilibrated with 40% acetonitrile with 0.1% trifluoroacetic acid (TFA).A 2ml aliquot (16mg protein) was injected and elution was performedisocratically with a mobile phase of 40% acetonitrile in water with 0.1%TFA concentration. The flow rate was set at 2ml/min and the chart speedwas set at 0.25cm/min. The chromatography was performed at roomtemperature. Five ml fractions were collected. Aliquots of each fractionwere lyophilized and assayed in triplicate for benzodiazepine (BZD)binding competing activity (BZD-BCA) and growth inhibitory activity.

Once the position of the BZD-BCA was known, (fraction 24) 49chromatographic runs as described above were made. The active fractionsfor all runs were pooled and lyophilized. About 65mg active of driedpowdered was obtained. This was called the TSK-250 fraction. Itcontained about 936 units of BZD-BCA activity.

Reverse Phase High Pressure Liquid Chromatography (HPLC) of TSK-250Fraction

TSK-250 fraction was dissolved (2mg/ml) in 0.1% TFA and furtherfractionated by reverse phase HPLC using a μ-Bondapak-C₁₈ column (78mmi.d.×30cm) at room temperature. The sample was applied isocratically andthe column was washed and equilibrated with 0.1% TFA. The flow rate wasset at 2ml/min and the chart speed was 0.25cm/min. Linear gradients wereused between the primary solvent 0.1% TFA and the secondary solventacetonitrile in 0.1% TFA. The gradient conditions were 0-28% in 20min.,then 28-42% in 140min, 42-52% in 10 min, and then 52-100% in 6 min. Allsolvents were HPLC grade. Four ml fractions were collected. Aliquots ofeach fraction were lyophilized and assayed in triplicate for BZD-BCA.Most of the activity eluted between 31-32% acetonitrile concentration.

Fractions 36 and 37 were pooled and diluted with 12ml of 0.1% TFA. Themixture was applied isocratically on a μ-Bondapak-C₁₈ column (3.9mmi.d.×30cm) at room temperature. The flow rate was 1ml/min and the chartspeed was 0.25cm/min. Again, linear gradients were used between theprimary solvent 0.1% TFA and the secondary solvent acetonotrile with0.1% TFA. The gradient conditions were as follows: 0-30% in 10min, then30-40% in 60min. Fractions were collected. Almost all of the activity(about 850 units) was eluted in fraction 6 at ˜31% acetonitrileconcentration. This fraction was called HPLC-C18 fraction and had about680μg protein.

Human EBZD was also purified substantially following the above describedprocedure with human brain tissue.

Preparation of Brain Synaptosome

Crude synaptic membrane fractions were prepared either from the brain ofSprague-Dawley rats weighing ˜200g or fresh bovine brain cortex asdescribed by Zuckin, et al., Proc. Natl. Acad. Sci. USA (1974)71:4802-4806. The synaptic membranes were washed three times with 50mMTris-HCl, pH 7.4 by repeated suspending in the buffer and pelleting bycentrifugation. The washed synaptosomal membranes were suspended in 50mMTris-HCl, ph 7.4 and stored at -20° C.

Assay for EBZD-Binding Competing Activity

The inhibition of the binding of ³ H-BZD to the washed synaptosome byvarious fractions or purified protein was used as an indicator ofbinding competing activity. The binding of ³ H-BZD to synaptosomalmembranes was performed in duplicate in 12×75mm disposable polypropylenetubes either in the absence or presence of 25μM β-carboline. The bindingmixture contined 20mM Tris-HCl, pH 7.4, synaptic membrane suspension(70-100μg protein), as desired various concentrations of test compound,1-5mM of ³ H-BZD, and 0.5% final concentration of dimethylsulfoxide in atotal volume of 0.1ml. After incubation for 30min at 4° C., 0.7ml of 10%cold polyethylene glycol (PEG 6000) in 0.1M Tris-HCl (pH 7.4) was addedto each tube. The suspension was immediately filtered under a vacuum onGF/B filter at 4° C. The filter was washed with 10ml of cold 50mMTris-HCl (pH 7.4) containing 1mg/ml BSA. The filters were transferred tocounting vials and 10ml of Aquassine (NEN) were added to each vial andradioactivity determined using a Beckman β-counter. The radioactivitybound in the presence of 25μM β-carboline (2-8% of total binding) wasconsidered to be nonspecific and data were corrected accordingly. Theprotein concentrations were determined by the method of Lowry, et al.,J. Biol. Chem. (1951) 193:265-275 using BSA as a standard.

Polyacrylamide Gel Electrophoresis (PAGE)

A 15cm resolving gel (0.75mm thick) of 15.6% polyacrylamidebis-acrylamide (30:0.8) containing 0.1M sodium phosphate, pH7.2, 0.1%SDS and 6M urea was used. The gel contained 10μl TEMED (Bio-Rad) per20ml of gel and was polymerized by using 1.0μl/ml gel of 20% ammoniumpersulfate. An upper gel of a 3.5% acrylamide solution using bufferconditions identical to those of resolving gel was poured on top of thelower gel. The comb was inserted into the top gel, leaving about 3mm ofupper gel. Forty μl of samples in 0.01M sodium phosphate pH 7.2, 7Murea, 1% SDS and 1% 2-mercaptoethanol, were boiled for 2min and quicklyapplied. The running buffer was 0.1M sodium phosphate, pH 7.2 containing0.1% SDS. The gel was run at 5V/cm at room temperature until thetracking dye reached the bottom of the gel. The gel was fixed in 50%methanol and 9 % acetic acid, stained with 0.1% Coomasie blue in thefixing solution, and destained with 50% methanol and 9% acetic acid.Following destaining, the gel was dried.

The PAGE method of Laemmli, Nature (1970) 227:680-685 was also usedemploying 15% resolving and 5% stacking gels. Protein on the gels wasdetected either by the silver staining method of Merril, et al., Science(1981) 211:1437-1439, or by Coomasie blue staining.

Iodination of Proteins

Proteins were labeled with ¹²⁵ I using the chloramine-T method asdescribed by Barridge, Methods Enzymol. (1978) 50:54-65, or by using ¹²⁵I-Bolton and Hunter reagent, Biochem. J. (1973) 133:529-539.

Antibody Production Against EBZD

Antisera to the purified EBZD from bovine brain were prepared in rats.Six-week-old Sprague-Dawley rats were primed with a total of 20μg ofpurified protein emulsified in Freund's complete adjuvant. Subsequentbooster inoculations were given at two-week intervals using 10μg ofprotein in Freund's incomplete adjuvant. Test bleeds were taken one weekafter each inoculation and screened for antibodies using theradioimmunoassay procedure outlined below. Antisera suitable for theassay were generally obtained after only two to three boosterinoculations.

Antibody sera was also prepared to the purified bovine brain EBZD inrabbits (New Zealand white weighing approximately 3kg) by a similarmethod as described for rats except 40μg and 20μg proteins were used,respectively, for the primary inoculation and for the boosterinjections.

Radioimmunoassay

Purified bovine brain factor was radioiodinated with Chloramine-T to aspecific activity of approximately 3×10¹⁰ cpm/μg and stored at 4° C. inTNEN buffer (20mM Tris-HCl, pH 7.4; 5mM EDTA; 150mM NaCl; 0.05% NP40;0.1% BSA).

For the radioimmunoassay, the following reagents were successively addedto polypropylene tubes (3ml capacity): 10μl of purified brain factorstandard or sample, 10μl of rat antiserum (diluted 1:30 in TNEN buffer)and 30μl of ¹²⁵ I-labeled bovine brain factor (˜5×10⁴ cpm). After a45min incubation at room temperature, 50μl of a 10% suspension ofheat-inactivated formalin-fixed S. aureus were added and the mixtureleft for an additional 30min. The immunoabsorbent was then centrifuged(15,000xg, 1min) through a cushion of n-butyl phthalate oil and theamount of radioactivity in the S. aureus pellet determined byγ-spectrometry. The radioactivity bound in the absence of antiserum wasconsidered to be nonspecific and data were corrected accordingly.

Tissue to be assayed for RIA reactive material was processed as follows.Fresh or frozen tissue (approximately 1g net weight) was added to 10mlof cold homogenization buffer (20mM Tris-HCl, pH 7.4; 5mM EDTA; 150mMNaCl; 0.2% NP40; 0.2mM phenylmethylsulfonyl fluoride; 100 kallikreininhibitor units of Trasylol per ml) and homogenized by three 15secbursts in a Polytron tissue homogenizer. The extract was removed andcleaned of debris by centrifugation for 30min at 100,000xg. Thesupernatant was then heated to 95° C. for 5min and centrifuged at lowspeed to remove precipitated protein. Serial 1:3 dilutions of theextract were made in TNEN buffer and used in the radioimmunoassay. Astandard curve was included with each set of assays and the amount ofimmunological reactive material estimated by direct comparison.

Peptide Cleavage by Various Proteases and Isolation of PeptideFragments.

Bovine and human EBZD were digested in 40μg of 0.1M Tris-acetate, pH8.0, with the endoproteinase Lys-C for 16h at 24° C. The ratio of EBZDto the enzyme was 10:1 on weight basis. The peptide fragments wereseparated by rpHPLC on a μ-Bondapak-C₁₈ column (Waters) using a linear2h gradient from 0.05% TFA in water to 60% acetonitrile containing0.045% TFA. The peptides were monitored and identified by theirabsorption at 214 nm, collected, lyophilized, and used for the assays.

RESULTS Purification and Certain Characteristics of Bovine Brain EBZD

The elution profile of EBZD of bovine brain from a column of Bio-SilTSK-250 was determined. The BZD binding competing activity emerged fromthe column as a single peak with a median size slightly smaller thanribonuclease (Mr-11,500). The result of chromatography of activefraction 24 from the TSK-250 column on a preparative reverse phasecolumn was that the activity eluted between 31-32% of acetonitrile.Rechromatography of pooled fractions 36 and 37 on an analytical reversephase column resulted in the elution of active protein as a symmetricalpeak. The purification is summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Purification of Bovine Brain EBZD                                                      Weight             Specific activity                                                                       Yield                                   Fraction (mg)       Units.sup.a                                                                           (units/mg)                                                                              (%)                                     ______________________________________                                        Crude    785.sup.b  1,010.sup.d                                                                           1.29      100                                     TSK-250   65.sup.c  936     14.4      92.7                                    (fraction 24)                                                                 HPLC-C.sub.18                                                                            .sup. 0.68.sup.c                                                                       850     1,250     84.2                                    ______________________________________                                         .sup.a Material needed for 40% competion of .sup.3 Hdiazepam binding to       crude synaptosomic membrane under the assay condition described above.        .sup.b Pooled material weighed directly.                                      .sup.c Calculated from absorption at 280 nm.                                  .sup.d Due to low specific activity, it was not possible to assay directl     at this stage. This number indicating total units recovered after TSK250      chromatography in fractions 24 and 25.                                   

A 969-fold purification with 84.2% yield was achieved in the three-stepprocess. The degree of inhibition increased with increasingconcentration of protein. However, the maximum inhibition was found tobe only 52% even at 8μM concentration of pure protein. The K_(i) valueswere found to be about 5μM, whether ³ H-diazepam or ⁸ H-R015-1788 wasused as a ligand.

The homogeneity of the purified protein was demonstrated by PAGE in thepresence of 6M urea. The molecular weight of the peptide was estimatedto be 10.5kDal. The analysis of the purified proteins (bovine or human)by SDS-PAGE method of Laemmli also gave a single band with an apparentM_(r) ˜7kDal. The purified proteins emerged as a single symmetrical peakfrom Bio-Sil TSK-250 column (60cm×7.5mm i.d.) using 40% acetonitrile inwater with 0.1% TFA as eluting solvent. The apparent molecular weightwas calculated to be about 11.5kDal by this gel permeationchromatography method. The purified human brain EBZD exhibited similarphysicochemical and biological properties.

Specific binding of ¹²⁵ I-labeled (either by Chloramine-T method or byBolton and Hunter method) purified protein to the synaptosomal membranewas not observed.

Distribution of EBZD

An RIA system was developed to quantitate EBZD in tissue extract, bodyfluids, and tissue culture cells. The displacement of ¹²⁵ I-labeled EBZDfrom bovine brain bound to rat antiserum against bovine EBZD by variouspeptides was determined. Bovine and human EBZD exhibited a similarpotency in competing with ¹²⁵ I-labeled bovine EBZD bound to ratantiserum prepared to the bovine EBZD protein indicating the presence ofquite similar immunogenic determinant(s) in both proteins. None of thepeptide fragments of bovine EBZD obtained by endoproteinase Lys-Cdigestion and purified by reverse phase HPLC showed anyimmunoreactivity. Thus, the immunogenic determinant of EBZD is notretained on any of the single peptide fragments generated byendoproteinase Lys-C. The distribution of EBZD in various tissues ofrabbit is presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        Distribution of EBZD in Various Rabbit Tissues                                               μg Equivalent/g                                             Tissue         Wet Tissue                                                     ______________________________________                                        Brain          3.80                                                           Kidney         3.42                                                           Salivary Gland 1.76                                                           Liver          1.52                                                           Stomach        1.27                                                           Colon          1.05                                                           Spleen         0.97                                                           Lung           0.84                                                           Heart          0.73                                                           Thymus         0.67                                                           Pancreas       0.50                                                           Thigh Muscle   0.37                                                           Thyroid        0.06                                                           Serum or Plasma                                                                              N.D.                                                           ______________________________________                                         N.D. = Not detectable.                                                        The details of RIA methods are given previously.                         

All the tissues tested except serum or plasma contained EBZD as detectedby RIA. Bovine brain, spleen, and thymus contained about 11.5, 7.6, and6.8 μg equivalent per g wet tissue, while human brain was found tocontain about 15.7 μg equivalent per g wet tissue. The concentration ofEBZD in rabbit brain was found to be much lower than that present inbovine or human brain. EBZD was also found in monkey brain. Theantiserum against bovine protein barely detected immunologically crossreacting material in rat and mouse brain. EBZD was also found to bepresent in human cerebrospinal fluid and ascites fluid. Human breastcarcinoma cells (MCF7), liver carcinoma cells (HEP2), neuroblastomacells (MTB14), glioneuroblastoma cells (CCL127), and glioblastoma cells(HTB10) were estimated to contain 9.1, 8.9, 8.6, 0.32, and 0.26 μgequivalent of EBZD per ml packed cells (˜10⁹ cells). One μg of EBZDcontains about 6×10¹³ molecules. Thus, MCF7, HEP2, and HTB14 cellscontain ˜5×10⁵ molecules of EBZD per cell. Thus, EBZD is widelydistributed in mammalian tissues and unlike DBI is not a brain specificpeptide. The wide tissue distribution would suggest a general ratherthan a tissue specific function for EBZD.

                  TABLE 3                                                         ______________________________________                                        Distribution of EBZD in Various Regions of Rabbit Brain                                       μg Equivalent/g                                            Tissue          Wet Tissue                                                    ______________________________________                                        Cerebrum        3.4                                                           Cerebellum      7.5                                                           Medulla Oblongata                                                                             5.3                                                           Pituitary       1.3                                                           Plfactory Bulb  2.6                                                           Pineal Body     17.4                                                          Choroid Plexus  26.7                                                          Pons            14.3                                                          Vega Nerve      1.7                                                           Optic Nerve     16.2                                                          Olfactory Tract 8.3                                                           ______________________________________                                         The EBZD concentrations were determined by RIA as described previously.  

Table 3 summarizes the distribution of EBZD in various regions of rabbitbrain as determined by RIA. All regions of brain possess EBZD-likematerial, however, substantial regional variation in EBZD concentrationwas noted. The highest amount was found in choroid plexus, and thelowest in pituitary among all the rabbit brain regions tested.

EBZD Analeptic Activity

Male New Zealand rabbits weighing between 2.3-2.6kg were used throughoutthis study. Icv injections were made by the direct puncture method asdescribed by Jacob et al., Neuropharmacol. (1972) 11:1-16 but usingslightly different coordinates. The animals are prepared 2 days prior tothe experiment by drilling a small hole (0.8mm diameter) in the skull(under pentobarbital anesthesia). The hole is located 1.0mm lateral tothe midline and 1.0mm rostral to the bregma. On the day of theexperiment, just prior to the injections, local anesthetic is sprayed onthe skin wound. A #26 needle is inserted vertically to the depth of 12mmfrom the surface of the skull. Proper positioning is indicated by theappearance of cerebral spinal fluid. The needle is withdrawn, filledwith drug solution, and reinserted to the same depth. Injections arealways in a volume of 10μl over a 30-60sec period and made with asyringe microburet. Controls consisted of animals given the same volumeof sterile saline. All drugs were dissolved in sterile isotonic saline,and are expressed as their free base.

The animals are generally placed in stanchions 2-3hr prior to theexperiment. Colonic temperatures are measured by rectal thermisterprobes connected to a YSI Scanning Telethermometer which is speciallywired to record onto a Leeds-Northrup Speedomax recorder. Theexperiments are carried out in a constant temperature room of 22.0±1.0°C.

Analeptic activity is indicated by a shortened time of recovery of therighting reflex. Rabbits given 25mg/kg iv of pentobarbital undergoanesthesia during which time it is possible to place them on their backswithout having them flip back to the normal upright position. The timeat which they could no longer be maintained on their backs wasconsidered as the recovery time of the righting reflex.

Behavior was evaluated by gross observation of various parameters,including pupillary dilation, increased motor activity, increasedrespiratory rate, etc. In addition, certain stereotypic responses, suchas compulsive gnawing or scratching, were carefully observed.

bEBZD

10μg

10min after injection animal starts continuous chewing response.Respiration increased from 90/min to 204/min. Increase in motoractivity. No hyperreactivity. No analgesia.

100μg

10min after injection respiration decreased from 98/min to 65/min.Compulsive chewing, and mild excitation. Excitation was short-lasting.No hyperreactivity. No analgesia.

200μg

4min after injection animal closes its eyes. Decreased respiration(108/min to 56/min). Loss of righting reflex 10min after injection.Animal was not in deep anesthesia. Righting reflex was regained 28minafter bEBZD administration. No analgesia.

Bests Rate Regulation in Cultured Aggregates of Embryonic Heart Cells

Cardiac ventricles from 8 day old chicken embryos were dissociated intosingle cells and put into rotation culture for 3 days. This methodproduces small, regular spheres of cells, approximately 150 microns indiameter. Cells within an aggregate are electrically coupled with eachother and beat synchronously, with a regular constant rhythm (Myrdal andDeHaan, J. Cell. Phys. (1983) 117:319-325). On the basis ofelectrophysiological characteristics, this preparation resembles adultmammalian Purkinji fibers, the major cardiac conducting system (Myrdaland DeHaan, The Initiation of the Heartbeat. Denis Noble, ed. 1975,Oxford University Press, London, p. 7). These aggregates wereequilibrated at 24° C. and treated with bEBZD for 24hr. In two separateexperiments, the beat rates of aggregates treated at doses of 100μg/mldiffered significantly from controls, as follows:

    ______________________________________                                        Experiment 1: (Beat rate measured as seconds/5                                interbeat intervals)                                                                     N     MEAN       STDEV  SE MEAN                                    ______________________________________                                        Untreated  14    11.48      1.44   0.38                                       Treated    14     7.94      2.04   0.55                                       ______________________________________                                        Experiment 2:                                                                            N     MEAN       STDEV  SE MEAN                                    ______________________________________                                        Untreated  13    24.59      3.18   0.88                                       Treated    12    19.33      2.37   0.68                                       ______________________________________                                    

In each experiment, by the Student's T test, the treated populationdiffers significantly from the untreated population. The probabilitythat these populations are the same is less than 0.0001 (p=0.0000).

Sequencing

The amino acid sequences of bovine and human EBZD were determined bymicrosequence analysis of peptides obtained from digests of bEBZD andhEBZD with (a) the endoproteinase Lysine C; (b) chymotrypsin; (c)Staphylococcal aureus V8; and (d) cyanogen bromide. The peptidefragments were purified by rpHPLC using volatile solvents. Aminoterminally blocked peptides were incubated in 12N HCl at ambienttemperature for 16hr. Samples were then dried by lyophilization. Thepeptides were subjected to automated repetitive Edman degradation in theModel 470A gas phase Protein Sequencer (Applied Biosystems, Inc.). Thephenylthiohydantoin amino acids were analyzed by rpHPLC.

Alternatively, the proteins (bovine and human EBZD) or peptides werehydrolyzed with 6N HCl at 105° C. for 16-20h at reduced pressure. Theresulting amino acids were converted to the phenylthiocarbamoylderivatives and analyzed by Pico TAG system (Waters Associates).Phenylthoicarbamoyl amino acids were detected by absorbance at 254nM.

Chemical Structure of EBZD

The amino acid compositions of bEBZD and hEBZD were determined afterhydrolysis with 6N HCl using an automatic amino acid analyzer. Allcommon amino acids except cysteine were present in these proteins. Theminimal molecular weight calculated from these data is approximately9,900 in agreement with that established by SDS-PAGE.

No N-terminal amino acid was detected, even when several cycles of Edmandegradation were performed, suggesting that the terminal amino groups ofbEBZD and hEBZD are blocked. The sequences were determined as describedin the Experimental section. The sequences of bEBZD and hEBZD are asfollows, with a comparison of the published sequence of DBI: ##STR5##Residues in the hEBZD amino acid sequences which differ from the bEBZDsequences are underlined.

A comparison of the amino acid sequences of hEBZD with the amino acidsequences of bEBZD indicates that human and bovine EBZD may differ fromeach other by only a few conservative substitutions. Five of six aminoacid substitutions are compatible with a single base change at the DNAlevel. These results establish that human and bovine EBZDs are highlyconserved structurally among different species.

Identification and Characterization of a cDNA Clone for a Factor fromBovine Brain.

Poly (A+) RNA isolated from bovine tissue was used as a template forcDNA synthesis. First strand cDNA synthesis was performed using oligo(dT) and avian myeloblastosis virus (AMV) reverse transcriptase; thesecond strand was synthesized using E. coli RNase H, DNA polymerase I,and DNA ligase (NAD+). The double-stranded cDNA was made blunt-ended byS₁ nuclease digestion followed by a fill-in reaction with the largefragment of E. coli DNA polymerase I. Terminal deoxynucleotidyltransferase was used to enzymatically add approximately 15 deoxyguanineresidues to the 3' ends of the cDNA. This G-tailed cDNA was thensize-fractionated on an A-50 column to eliminate both cDNAs smaller than500 base pairs, as well as unincorporated deoxyguanine residues. PooledcDNA was then concentrated by ethanol precipitation in the presence ofEcoRI digested λgt.10 DNA.

Ligation of the G-tailed cDNA to EcoRI cut λgt.10 was achieved via anovel method utilizing a single-stranded oligonucleotide linker whichcontained 12 deoxycytosines at the 3' end and a sequence (AATT)complementary to the single-stranded overhang of EcoRI-cleaved DNA atthe 5' end. After in vitro packaging of the ligated DNA, recombinantphage were introduced into the E. coli strain C600rk^(-mk) ⁺ Hfl, whichundergoes cell lysis when infected by recombinant, but not wild type,phage. DNA from plaques were thereby screened in duplicate using aradiolabeled synthetic oligonucleotide, 17 nucleotides in length, thesequence of which was predicted by 6 consecutive amino acids (KWDAWN)found in the bovine EBZD; due to codon ambiguity, MSl was synthesized asa 32-fold degenerate pool of oligonucleotides. Screening with MS1produced a positive clone which was plaque purified and shown to containa 1.8Kb insert. This insert was subcloned into the plasmid pEMBL and isdesignated pMP1. After restriction mapping of the insert, appropriatefragments were then further subcloned into strains of M13 and sequencedby the dideoxy method of Sanger and Coulsen.

The cDNA contained in pMP1included a DNA sequence approximately 300 basepairs from the 5' end of the insert which was nearly identical to aspecies of MS1. Furthermore, the nucleotide sequence of pMP1 predictedan amino acid sequence similar, but not identical, to that determinedfor the sequenced bovine EBZD. That these two proteins are closelyrelated is suggested by the striking conservation of amino acidsequence: by deleting three amino acids from the sequenced EBZD, it ispossible to align the remaining amino acids with the sequence of pMP1 sothat 43% of the amino acids are conserved. In addition, a number ofalterations comprise conservative substitutions. (See earlier chart.)

The finding of pMP1 demonstrates that the bovine EBZD is a member of afamily of related genes which may encode proteins of similar biologicalactivity. Finally, it should be noted that pMP1 encodes a protein largerthan the sequenced bovine brain factor since an open reading frameextends both upstream and downstream from the known terminal amino acidsof the latter protein.

Expression of the EBZD Gene

The above procedure was repeated using bovine spleen. The cDNA obtainedwas a 0.6kbp insert which was cloned into pEMBL to give pEBZD.Sequencing provided a coding sequence having homology to pMP1. The DNAfragment encoding the bovine endozepine (bEBZD) was excised from thecDNA plasmid, EBZD, by a combination of DraI and NaeI digestion. Thefragment was purified by gel electrophoresis and inserted into anexpression vector, pSM1,2, at the StuI site. The pSM1,2 expressingvector was developed by splicing the replication origin and β-lactamasegene of pBR322 (Bolivar et al., Gene (1977) 2:95), the lac promoter andribosomal binding sites of pTR213 (Roberts et al., Proc. Natl. Acad.Sci. USA (1979) 76:760-764) to the i-Z fused gene of pLG300 (Guarente etal., Cell (1980) 1 20:543-553). The resulting plasmid can express theforeign gene inserted at the cloning sites such as the StuI site as afused protein. In this construction the entire coding sequence for EBZDwas fused in frame to the DNA encoding the first 21 amino acids of theCro protein. The plasmid pSB108 has the insert in the right orientation;the plasmid pSB103 has the insert in the wrong orientation; the plasmidpSB125 contains no insert. The E. coli HB101 clones harboring variousplasmids were grown up to log phase and induced by addition of IPTG to afinal concentration of 1mM at 37° C. for 2hr with shaking. The cellswere harvested by centrifugation and lysed by the lysozyme-detergenttreatment. Basically, the lysate can be divided into supernatant(cytosol) and pellet (inclusion body) and assayed by immunochemicaltechniques.

The levels of expressed EBZD fused protein were measured by competitiveradioimmunoassay. The results indicate that only the E. coli clonecontaining the EBZD expression vector with the insert in the rightorientation (pSB108) produces high levels of EBZD polypeptide. Mostcro-EBZD was found in the supernatant fraction (cytosol). Only adetectable amount was found in the pellet fraction (inclusion body). Onthe other hand, no cro-EBZD was detected in the lysate of either thepSB103 or pSB125 clone. It is estimated that 0.5mg of the cro-EBZD wasproduced by one liter of cell culture after induction by IPTG.

Purification of Brain Factor from Bovine Brain Gel PermeationChromatography

A Bio-Sil TSK-250 preparative column (60×2.1 cm) was attached to a highpressure liquid chromatography (HPLC) system (Waters Associates). Thecrude fraction was dissolved in 40% acetonitrile in water with 0.1%trifluoroacetic acid (TFA) at a concentration of 8mg/ml. The column wasequilibrated with 40% acetonitrile with 0.1% TFA. A 2ml aliquot (16mgprotein) was injected and elution was performed isocratically with amobile phase of 40% acetonitrile in water with a final 0.1% TFAconcentration. Once the position of BF was known (fraction 23), a fewhundred runs as described previously were made. The procedure formonitoring the fractions was the cell growth modulatory assay describedbelow. The apparent molecular weight of the brain factor (GIA) wascalculated to be about 18kDal from this permeation chromatography.

Reverse Phase High Pressure Liquid Chromatography of TSK-250 Fractions

TSK-250 fraction 23 from 15 runs were pooled (volume 75ml). The pooledmaterial was diluted two-fold with 0.1% TFA in water. This mixture wasinjected isocratically on a μ-Bondapak-C18 column (78mm i.d.×300mm)previously equilibrated with 0.1% TFA in water (0.1% TFA) at ambienttemperature. Linear gradients were used between the primary solvent 0.1%TFA and the secondary solvent acetonitrile with 0.1% TFA. The gradientconditions were 0-28% in 20min. 28-42% in 140min, 42-52% in 10min, andthen 52-100% in 6min. All solvents were HPLC grade. Four ml fractionswere collected. Aliquots of each fraction were dried with 50μg BSA andassayed for GIA. Two peaks of GIA were observed. The first activity (α)eluted between 32-33.5% acetonitrile concentration whereas the secondpeak (β) eluted between 34.5-36% acetonitrile concentration. Furtherpurification of α activity was performed as follows:

Active fractions 30 and 31 from 10 runs were pooled and diluted two-foldwith 0.1% TFA. The diluted sample was applied isocratically onμ-Bondapak column (78×300mm) and column was washed and equilibrated with0.1%TFA. The flow rate was 1ml/min and the chart speed was 0.1cm/min.Again, linear gradients were used between the primary solvent 0.1% TFAand the secondary solvent acetonitrile having a concentration of 0.1%TFA. The gradient conditions were 0-30% in 40min, 30-38% in 240min, and38-100% in 20min. The first 12 fractions were 6ml and then 4ml fractionswere collected. An aliquot was assayed for GIA. The activity elutedbetween 32-33.5% acetonitrile concentration.

Fractions 30-32 were pooled. Twelve ml of 0.1% TFA were added to thepooled fractions. The mixture (24ml) was isocratically applied onto ananalytical μ-Bondapak C18 column (3.9×300mm), equilibrated with 0.1%TFA. The flow rate and chart speed were 0.4ml/min and 0.1cm/min,respectively. Again, linear gradients were used between the primarysolvent 0.1% TFA and the secondary solvent acetonitrile having aconcentration of 0.1% TFA. The gradient Conditions were 0-30% in 25min,30-38% in 200min, and 38-100% in 25min. Two ml fractions were collected.An aliquot of the fractions were assayed for GIA. The activity elutedfrom the analytical C18 column at about 34% acetonitrile concentration.

Fractions 29-31 were pooled and diluted two-fold with 0.1% TFA andapplied on a μ-Bondapak C18 column (3.9×300mm). Linear gradients betweenthe primary solvent 0.1% TFA and the secondary solvent n-propanol havinga concentration of 0.1% TFA were used. The gradient conditions were0-18% in 40min, 18-27% in 225min, and 27-35% in 20min. The flow rate was0.4ml/min and the chart speed was set at 0.1cm/min. Fractions werecollected and aliquots assayed for GIA. The activity eluted at about 23%n-propanol concentration.

Fractions 28 and 29 were pooled and diluted five-fold with 0.1% TFA andrechromatographed on a μ-Bondapak C18 column (319×300mm) using n-propanohaving a concentration of 0.1% TFA as a secondary solvent. Thechromatographic and gradient conditions were the same as describedimmediately above. The first eight fractions were 6ml and then 1.6mlfractions were collected. Aliquots of each fraction were assayed forGIA. Most of the activity appeared in fractions 15 to 17. Fraction 15 to17 contained about 15μg protein and approximately 230×10³ units of GIA.This fraction was termed HPLC-C18⁵ fraction. The final purified fractionhad a specific activity of 15.33×10³ units per μg protein using CCL64 astest cells.

The following Table 4 summarizes the results.

                  TABLE 4                                                         ______________________________________                                        Purification of Bovine Brain Factor                                                    Weight   Units*   Specific Activity                                                                         Yield                                  Fraction (mg)     (×10.sup.-3)                                                                     (Units/mg × 10.sup.-3)                                                              (%)                                    ______________________________________                                        Crude    3,680    1,551    0.421       100                                    TSK-250  630      2,760    4.38        181                                    HPLC-C18.sup.5                                                                         0.015      230    15,333.00   14.8                                   ______________________________________                                         *Material needed fcr 50% inhibition of .sup.125 Ideoxy uridine                incorporation into DNA of CCL64 cells.                                   

Cell Growth Modulatory Assay Using ¹²⁵¹ -Deoxyuridine Incorporation intoDNA

The assays were performed in Nunc 96 well plates (Kamstrupvej 90.DK-4,000, Roskilde, Denmark). Human lung carcinoma cells (A549) or minklung cells (CCL64) were used as test cells. 3.5×10³ cells in 50 μl ofDulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum(FCS) and penicillin/streptomycin (P/S) (0.57 mg/ml each) and glutaminewere introduced to all wells except peripheral wells. The peripheralwells received 50 μl PBS and the plates were incubated at 37° C. Thetest samples were suspended in DMEM with 10% FCS, P/S, and glutamine fortriplicate testing. After 4 hours, 50 μl of test samples were added toeach test well, while control wells received only 50 μl of medium.Plates were incubated at 37° C. for 3 days. On day 4, 100 μl of asolution of ¹²⁵ I-iodo-2'-deoxyuridine [(4Ci/mg-0.5 mCi/ml 1.0μ)](1.0 μlisotope/ml in DMEM containing 10% FCS, P/S, glutamine) were added toeach well and plates incubated at 37° C. On day 5, the medium wasaspirated from the wells, washed 1x with 200 μl PBS. Then, 200μ ethanolwere added to each well, plates were incubated for 10 minutes andmethanol removed by aspiration. Sodium hydroxide (200 μl, 1M) was addedto each well, the plates were incubated for 30 min. at 37° C. and thensodium hydroxide was removed with Titertek plugs (Flow Labs). The plugswere transferred into 12×75 mm plastic tubes and counted in a gammacounter in order to quantitate the radioactivity.

Soft Agar Colony Inhibition Assay

A 2.0 ml base layer of 0.5% agar (Agar Noble; Difco Laboratories,Detroit, Mich.) in DMEM containing 10% calf serum was added to 60 mmCostar tissue culture dishes. A 2.0 ml overlay of 0.3% agar containingthe same medium-calf serum mixture, 1.0×10⁴ A549 Ag3 (soft agar growingclone 3) cells and the sample to be tested at various concentrations induplicate were added. The plates were incubated at 37° C. in ahumidified atmosphere of 5% CO₂ in air and refed after 7 days byaddition of 2.0 ml of 0.3% agar containing appropriate supplements.Colonies were measured unfixed, unstained and the number of coloniesgreater than 6 cells per 5 low power random fields were scored after 7and 14 days.

Plating Efficiency Assay

The assay was performed in 6 well Falcon plates (9.6cm² area/well). A549cells (200) were plated in each well in 1ml of DEM with 10% FCD, P/S andglutamide. Immediately following plating, various concentrations of testmaterial in 1.0ml of medium in duplicate were added to wells. Thecontrol wells received only 1.0ml of medium without any test material.Plates were incubated at 37° C. in a humidified atmosphere of 5% CO₂ inair for 10 days, medium aspirated, 1.0ml of 0.2% methylene blue in 50%methanol was added at each well and allowed to stand for 20min, stainremoved, each well washed 2× with 0.1ml water and air dried. The sizeand number of colonies were quantitated.

Biological Properties

A 50% inhibition of DNA synthesis was observed at 98μg/ml, 21μg/ml and95ng/ml of crude fraction, TSK-250 fraction and the final pure protein,respectively. Thus, a 50% DNA synthesis inhibition in A549 human lungCarcinoma cell was seen at approximately 9nM concentration of the pureprotein.

bBF also inhibited the anchorage independent growth of human carcinomalung cells A549 on agar (Ag). A 50% reduction in colony formation insoft agar was seen at about 76ng/ml of bBF. The plating efficiency ofA549 cells was markedly inhibited by the protein. At 13ng/ml (1.24nM)concentration of protein, the plating efficiency was only 50% comparedto that of the control. No colonies of A549 cells were seen in thepresence of approximately 80ng/ml bBF.

When A549 cells were grown in the absence and presence of variousconcentrations of bBF, and cell growth monitored by direct cell count,it was found that bBF inhibited cell growth in a dose dependent manner.Thus, the extent of ¹²⁵ I-deoxyuridine incorporation into DNA is a goodmeasure of cell growth.

bBF inhibited the growth of various clones of A549 cells, human melanomacells (A375 Ag) and human breast carcinoma cells (MCF-7). The growth ofhuman foreskin fibroblast (WI-26) was stimulated by bBF. The brainfactor was also growth stimulatory to murine fibroblastic cell line3T3-A31.

Effect on Human T Lymphocyte Proliferative and Cytotoxic Responses toAlloantigens

A micromethod for generating and assaying allogeneically inducedproliferative and cytotoxic human lymphocytes was used to study theeffect of brain factor on human T-cell functions (Zarling et al.,Transplantation (1976) 21:468-476). Periphera blood lymphocytes (PBL)were isolated from heparinized blood of normal individuals byFicoll-Hypaque centrifugation. The PBL were then washed 3× withphosphate buffered saline (PBS) and were suspended at a concentration of1×10⁶ PBL/ml in RPM1 1640 medium (Gibco, Grand Isle, NY) supplementedwith 10% heat-inactivated pooled normal human serum (HS). Then 0.1ml ofthese PBL (referred to as "responding cells") was added to each of tenreplicate round-bottomed wells of 96-well plates followed by theaddition of 0.05ml medium alone or 0.05ml medium containing 2×105X-irradiated (2500 Rad) allogeneic cells (referred to as "stimulatingcells") and 0.05ml medium alone or medium containing variousconcentrations of brain factor (resulting in 0.9 to 75 unites brainfactor/well). The cells were incubated at 37° C. in a 5% CO₂ incubatorand on days 2 and 5 0.05ml medium was removed from each well followed bythe addition of 0.05ml medium containing the original concentration ofbrain factor.

To determine proliferative responses, on day 6 the contents of wellsfrom each group were pooled and 0.1ml was added to each of fourreplicate wells of 96-well plates followed by the addition of one μCi ³H-thymidine (³ H-TdR, New Engand Nuclear, Boston, MA) in a volume of0.025ml. Six hours later the contents of wells were harvested on glassfilter strips using a multiple well harvestor, the strips weretransferred to vials containing scintillation fluid, and ³ H-TdRincorporation was determined by counting in a β counter.

To determine the effect on the generation of cytotoxic T lymphocytes(CTL), the remaining lymphocytes pooled from wells on day 6 were addedto three replicate round-bottomed wells of 96-well plates (0.15ml/well)and 0.15ml of serial four-fold dilutions was also added to each of threereplicate wells. To prepare ⁵¹ Cr labeled target cells, 1.5×106lymphoblastoid cell line (LCL) cells, generated from the donors of theallogeneic stimulating cells, were labeled with 500μCi ⁵¹ Cr (Na₂ CrO₄),New England Nuclear, Boston, MA) for 1hr at 37° C. followed by washingthe target cells 3× with medium containing 15% heat-inactivated fetalcalf serum (FCS) and were resuspended in this medium at cells/ml. Then0.05ml target cells was added to each well containing effector cells andto wells containing the medium alone (to determine spontaneous ⁵¹ Crrelease) or detergent alone (to determine ⁵¹ Cr release) and the plateswere incubated for 6hr at 37° C. Then a constant aliquot of supernatantwas removed from each well and transferred to tubes for counting in agamma counter. The percent specific ⁵¹ Cr release was determined asfollows: ##EQU1##

The following Table 5 indicates the results:

                  TABLE 5                                                         ______________________________________                                        Effect of Brain Factor on Alloantigen-induced                                 Proliferative Response of Human T-Lymphocytes                                 Respond-                                                                             Stimulating                                                                             Brain Factor                                                                             CPM .sup.3 H-TdR                                                                       Percent                                  ing Cells                                                                            Cells     Units/Well Incorporated                                                                           Reduction                                ______________________________________                                        V      0         0            207                                             V      Cx        0          103,690                                           V      Cx        75         70,140   33                                       V      Cx        25         64,507   38                                       V      Cx        8          78,287   25                                       V      Cx        2.7        87,095   16                                       V      Cx        0.9        99,552    4                                       ______________________________________                                         Peripheral blood lymphocytes (PBL) were isolated from heparinized blood o     normal individual V and were stimulated with Xirradiated (2500 Rad)           allogenic PBL from individual C (Cx); and 6 days later, .sup.3 Hthymidine     (.sup.3 HTdR) incorporation was determined as detailed above. CPM = count     per minute.                                                                   Similar effects of brain factor were observed on Tcell proliferative          response of PBL of all other donors tested. A more marked degree of           inhibition by brain factor was observed on the generation of human            cytotoxic Tlymphocytes.                                                       Approximately 16 times more effector cells incubated with 75 units of         brain factor are required to cause 42% .sup. 51 Cr release as the number      of untreated effector cells to cause this same amount of .sup.51 Cr           release.                                                                 

It is evident from the above results that the subject compounds can beused in a wide variety of ways, both in vitro and in vivo. The subjectpolypeptides and antibodies can be used diagnostically for determining,intracellularly or extracellularly, in physiological fluids, e.g.,blood, serum, plasma, urine, or cerebrospinal fluid, the presence ofsuch polypeptides by recognized diagnostic assays, for determination ofthe amount of the polypeptide formed by cells in tissue. Also, thesubject compounds can be used for the detection of receptors for thepolypeptides. In addition, the subject compounds can be used formodulating the rate of growth of tumor cells, both in the presence andabsence of normal cells, as immunomodulators or for modifyingphysiological functions associated with the diazepam receptors orGABA-ergic receptors. Thus, the brain factor compounds derivable frombrain tissue and fragments thereof can be used in conjunction with otheradditives for removing tumor cells from a mixture of normal and tumorcells, such as in bone marrow, tumors, e.g., melanomas, sarcomas, orcarcinomas, for surgical or post-surgical treatments, or the like. Thebrain factor compounds can also be used for modulating the proliferationof T-cells. The EBZD compounds may be used in modulating the activity ofthe EBZD receptors.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A DNA expression construct comprising a DNAsequence encoding a first polypeptide sequence having at least oneepitopic site cross reactive with a second polypeptide found inmammalian brain capable of specifically binding to the diazepam receptorin vitro, said first polypeptide including substantially the followingsequence or a physiologically active fragment of at least about 30 aminoacids thereof: ##STR6## where plurality of amino acids at a siteindicates that any amino acid at that site may be substituted by anyother amino acid at that site and an asterisk (*) intends a bond may beemployed at that site, said DNA sequence joined in proper orientation toat least one of a promoter other than the wild-type promoter, aterminator other than the wild-type terminator, or a replication systemother than the wild-type replication system.
 2. A DNA expressionconstruct according to claim 1, wherein said replication system,promoter and terminator are functional in a eukaryotic host.
 3. A DNAexpression construct comprising a DNA sequence encoding an activepolypeptide, said polypeptide characterized by eluting in the 20% to 26%n-propanol fraction in aqueous 0.1M trifluoroacetic acid-n-propanol,being of from about 8 to 18 kDal, inhibiting tumor cell or T-cell growthand being found in mammalian brains, or a physiologically activefragment thereof, said DNA sequence joined in proper orientation to atleast one of a promoter other than the wild-type promoter, a terminatorother than the wild-type terminator, or a replication system other thanthe wild-type replication system.
 4. A DNA expression constructaccording to claim 3, wherein said replication system, promoter andterminator are functional in a prokaryotic host.
 5. A DNA expressionconstruct comprising a DNA sequence encoding a polypeptide havingsubstantially the same amino acid composition as membrane protein as setforth in FIG. 1 or a fragment thereof, said fragment beginning atmethionine 37, 68 or 69, said DNA sequence joined in proper orientationto at least one of a promoter other than the wild-type promoter, aterminator other than the wild-type terminator, or a replication systemother than the wild-type replication system.
 6. A DNA expressionconstruct according to claim 5, wherein said replication system,promoter and terminator are functional in a prokaryotic host.